1)PCR

Material	Volume(μL)
2× Mix	20
Primer F (10 μM)	1
Primer R (10 μM)	1
DNA template	1
ddH2O	17
Total	40

 PCR instrument program settings：

Step	Temperature (℃)	Time
1	95	3 min
2	95	30 sec
3	55	30 sec
4	72	1 min
5	72	10 min
6	4	forever

PS:The step2 and step4 cycle 30 times 

 

2)Gel electrophoresis

a.Materiel

a)Electrophoresis chamber

b)Gel caster

c)Comb

d)Power supply and electrodes

e)Pipettes

f)Gel

Material	Mass/volume
Agarose	0.5 g
1xTAE	50 mL
GelRed	5 μL

 PS: TAE components: Tris base, acetic acid,and EDTA

g)Gox1 DNA samples

h)Trans15K DNA marker

i)6X Gel loading dye, purple 

b.Procedure

a)Weigh 0.5-gram agarose

b) Add it into the prepared 50mL TAE buffer in a conical flask

c) Heat it in the oven with medium or high heat

d) Shake it for a while after the first heating 

e) Heat it again

f) Wait until it cools down. Place a comb and a base in the electrophoresis device and pour the solution into it. 

g) Wait until it turns solid.

 

Material	Volume(μL)
E.coli	3 mL
SP1 buffer	250 μL
SP2 buffer	250 μL
SP3 buffer	250 μL
Wash buffer	1000 μL
Elution buffer	40 μL

Procedure

Extract plasmids from pET-28a bacterial fluid

1) Add 1mL E.coli into bacterial fluid

2) Centrifugate with 12000xg for 5 minutes to collect bacteria

3) After centrifugation, pour the waste fluid

4) Pour 2mL E.coli into the bacterial fluid again

5) Centrifugate again(same as previous)

6)Add 250μL SP1 buffer, then use a pipette to blow the fluids until the solids dissolve into the fluids

7) Add 250μL SP2 buffer, reverse the tube upside and downside for about ten times (to release protein and degrade RNA)

8) Leave it and wait for 2 minutes 

9) Add 350μL SP3 buffer(to precipitate protein), then reverse it again (same as previous)

10) If we could see the protein on the surface of the liquids, centrifugate it with 12000xg for 10 minutes.

11) After centrifugation, take the supernatant fluid and centrifugate it with 8000xg for 30 seconds

12) Add 500μL wash buffer and centrifugate it with 9000xg for 30 seconds

13) Add 500μL wash buffer again and centrifugate for 1 minute with 9000 xg(to centrifugate all wash buffer) 

14) Then add 40μL elution buffer, leave it and wait for 2 minutes

15) Centrifugate it with 9000xg for 1 minute and test the concentration of the plasmid.

 

Restriction enzyme digestion

Material	Volume(μL)
plasmid (pET28a)	30
NheI	1
XhoI	1
Buffer (10x) (Cutsmart)	5
ddH2O	13
Total	50

 

Material	Volume(μL)
DNA (Gox1)	25
NheI	1
XhoI	1
Buffer (10x) (Cutsmart)	5
ddH2O	18
Total	50

Placed them in a 37 ℃ metal bath for 1 hour.

 

Ligation

Material	Volume(μL)
pET28a Plasmid (digested)	10
GOX1 (digested)	7
T4 ligase	1
T4 ligase buffer	2
Total	20

Procedure:

1) Add 10μL pET28a Plasmid, 7μL target gene, 1μL T4 ligase and 2μL T4 ligase buffer into a new tube

2) Put the tube into the laboratory 16℃ metal bath for 30 minutes

3) Put it into the fridge

 

1) Mix 50μL Competent Cells(DH5α/BL21) with 5μL plasmid

2) Put it on the ice and wait for 30 minutes

3)Heat Shock: Transfer the mixture to a water bath or heat block set at 42°C for 90 seconds.

4) Put it on the ice again for 3 minutes

5) Add 900μL LB, and shake it with a shaker for 1 hour at 220rpm, 37°C

6) Centrifuge 9000rpm for 1 minute.

7) Spread it on the K+ flat and keep at 37°C overnight.

 

Type	Volume/Mass
Tryptone	5 g (10 g/L)
Yeast Extract	2.5 g (5 g/L)
NaCl	5 g (10 g/L)
ddH2O	500 mL
Agar(solid LB only)	7.5 g (1.5%)
Kanamycin	Working concentration 50 μg/mL
Total volume: 500 mL(+50 μL)

1) Put 1mL pET28a-GOX1 (BL21) strain into 400mL LB liquid medium, then add 45μL kanamycin.

2) Add 400μL IPTG solution (1M).

3) Put it into the shaker for 16h, 16 degrees.

 

1） The 4T1 cells are brought from Beyotime.

2） Put the cells into a water bath (37 degrees) as soon as it is received.

3） Transfer the cell suspension to a 1.5 mL tube, then put them into a centrifuge. (500g, 5min) 

4） Aspirate the supernatant, then resuspend it in a fresh complete culture medium and transfer it to culture vessels. 

5） Put it in a CO₂ incubator.

 

His A

Material	Mass
Na2HPO4 (40 mM)	1.42 g
NaCl (500 mM)	14.6 g
Imidazole (20 mM)	0.68 g
HCl (6 M)	Adjust the pH to 7.4
Add water to a constant volume of 0.5 L

His B

Material	Mass
Na2HPO4 (20 mM)	0.71 g
NaCl (500 mM)	7.3 g
Imidazole (500 mM)	8.5 g
HCl (6 M)	Adjust the pH to 7.4
Add water to a constant volume of 0.25 L

1)Protein purification

a.  

a) Take IPTG(1mM)-induced BL21(with pET28a-Gox1) bacterial solution and divide them into 6 centrifugal tubes

b)Centrifugate it with 5000rpm for 10 minutes to collect thallus, then discard the supernatant fluid

c) Pour 40mL bacterial fluid and repeat step (b) again

d) Pour 10mL bacterial fluid and repeat step (b) again

e) Add 15mL His-A buffer into the tubes

f) Use a pipette to blow the fluid for about 15 minutes. It might need to be put in the ice for several seconds after a period of blowing

g) Put the tube into the insulation bucket with ice, and make sure it is fixedly placed

h)Use ultrasonic cell crusher to crush it. (500W power, work 3s & stop 3s, 15 minutes)

b. 

Use centrifugation to collect supernatant fluid

a)Collect the fluid after ultrasonic crash and centrifugate it at 12000 rpm for 20 minutes, under the temperature of 4℃. 

b)Repeat the centrifugation

c) Collect the supernatant fluid, and the fluid is enzyme-containing crude suspension

 

2) Nickel column affinity purification

a) Collect the bacterial fluid into collection tubes and keep a little amount as a sample(for contrast)

b) Balance the nickel column with His-A buffer twice

c) Add the protein solution into the nickel column, and wait until it flows to the bottom

d) Add 10mL His-B buffer to elute protein(Keep it at 4℃)

e) Use 5mL of 20% ethanol to wash the column

f) Transfer the fluid to ultrafiltration centrifugal tubes

g) Centrifugate the tubes and test the concentration with the micro detector

 

 

Material	Volume
Lysate solution	15 mL
Working solution	174 μL
Solution C	6 μL
Loading	20 μL

Procedure:

1)Add 15mL bacteria solution and 15mL lysate solution

2)Use the extract from the glucose oxidase activity test kit to resuspend

3)Put it on ice (ice needs to be compacted), then use ultrasonic cell crusher to crush it at 500W power, work 3s & stop 3s, 15 minutes

4)Make the working solution(15mLA + 3mLB), then add 174μL of working solution and 6μL solution c.

5)Gel electrophoresis

6)Add 20μL loading. Add 5,10,20,40,60,80 μL samples to be tested respectively

7)80μL does not have purified cracked protein and washed protein with His buffer.

8)Place at 37 ℃ and measure at 5 minutes and 2 hours

9)Microporous template inspection system (450 nm, detection of light absorption value)

 

1)The protein solution is centrifuged in a concentration tube (12000 rpm for 5min).The machine should be cooled to 4°C before centrifugation.

2) Add 20μL protein loading to 80μL protein solution. Put it into boiling water for 10min.

3)Take 10μL of the solution, and used the micro detector to measure its concentration.

4)Gel electrophoresis (90V for 30 min, then 120V for 30min)

5)Dye with BBI Coomassie brilliant blue, then place in the microwave on medium heat for three minutes

6)Coomassie brilliant blue eluent

Material	Volume
Acetic acid	100 mL
Ethyl alcohol	400 mL
ddH2O	500 mL

1） Put cells in a 96-well plate at a density of 100,000/cm².

2） Data: RSL/1μM: pET28a-Gox1/ 16 32 64 μg/mL

3） Incubate the solution for 24 hours, then add 10μL CCK-8

4） Place the cells in a cell culture incubator at 37 degrees for 1 hour.

5） Measure the absorbance at 450nm.

 

1) Put cells in a 6-well plate at a density of 100,000/cm².

2) Data: RSL/1μM: pET28a-Gox1/ 16 32 64 μg/mL

3) Incubate the solution for 24 hours.

4) Flush with PBS once.

5) Add 1mL of reagent 1, then resuspend the cells.

6) Freeze-thaw with liquid nitrogen 2-3 times, then put it into a centrifuge. (12000 rpm, 10min)

7) Preheat the reagent 2 to 37 degrees. (30 min)

8) Prepare 1 mg/mL standard solution into 300µg/mL, 200µg/mL, 100µg/mL, 50µg/mL and 25µg/mL standard solution.

9) Add 20μL of standard, 140μL of reagent 2 and 40μL of reagent 3.

10) Homogenize the reagent, then measure the absorbance at 405nm.